Innovative orthogonal two-dimensional reversed-phase liquid chromatography × supercritical fluid chromatography with a phenyl/tetrazole stationary phase for the preparative isolation of diarylheptanoids

In this investigation, we successfully isolated and purified natural diarylheptanoids using an orthogonal offline two-dimensional RPLC x SFC approach, employing only the phenyl/tetrazole stationary phase. First, a styrenedivinylbenzene matrix medium pretreatment liquid chromatography system effectively processed chlorophyllcontaining plant extract solution with a recovery rate of 33.8 %, obviating the need for concentration steps. Subsequently, an offline two-dimensional RPLC x SFC employing only the phenyl/tetrazole stationary phase achieved a remarkable 96.38 % orthogonality and was established and utilized in the preparative separation and purification of natural products. Finally, the constructed single stationary phase highly orthogonal RPLC x SFC system was successfully applied in the preparative separation and purification of natural diarylheptanoids from the Saxifraga tangutica target fraction and yielded four diarylheptanoids with purities exceeding 95 %.